JP2003506090A - Unicellular or multicellular organisms for producing riboflavin - Google Patents

Abstract

  (57) [Summary] The present invention relates to a single cell or multiple cells for biotechnologically producing riboflavin, wherein the enzymatic activity for NAD (P) H-formation is higher than the enzymatic activity for NAD (P) H-formation of wild-type ATCC10895 of Acibia gossypii. It relates to cellular organisms, especially microorganisms.

Description

Detailed Description of the Invention

[0001]   The present invention relates to unicellular or multicellular organisms for producing riboflavin.

Vitamin B2, also called riboflavin, is essential for humans and animals. During vitamin B2 deficiency, inflammation of the oral and throat mucous membranes, cracks in the mouth, itching and inflammation in skin wrinkles, especially skin disorders, conjunctivitis, loss of vision and corneal haze appear. Growth arrest and weight loss can occur in mammals and children. Therefore, vitamin B2 has economic importance especially as a vitamin preparation when vitamins are deficient and as a feed additive. In addition, it is used as a food color, for example in mayonnaise, ice cream, pudding and the like.

Riboflavin is produced chemically or microbiologically. In chemical production processes, riboflavin is usually obtained as a pure end product in a multi-step process, which requires the use of relatively expensive starting materials, for example D-ribose.

Another alternative method for the chemical synthesis of riboflavin is to produce this substance by microorganisms. Microbial production of riboflavin is particularly suitable when high purity of this material is not required. This is the case, for example, especially when riboflavin should be used as an additive in feed products. If this material can be obtained in one step, microbial production of riboflavin is preferred. In addition, renewable raw materials, such as vegetable oils, can be used as starting materials for the microbial synthesis.

The production of riboflavin by fermentation of fungi such as Ashbya gossypii or Elemothecium ashbyi is known (The Merck Index, Windholz et al., Merck & Co., published, 1183). P. 198
3, A. Bacher, F. Lingens, Augen. Chem. 1969, p393) as well as yeasts such as Candida or Saccharomyces and bacteria such as Clostridium,
Bacillus and Corynebacterium are suitable for riboflavin production.

Further, a method using yeast Candida famata is described in US05
231007.

A riboflavin overproducing bacterial strain is described for example in EP405370, where this strain was obtained by transformation of the riboflavin biosynthesis gene from Bacillus subtilis. However, this prokaryotic gene is present in eukaryotes such as Saccharomyces cerevisiae.
siae) or Ashivia gossypii for recombinant riboflavin production. Thus, according to WO 93/03183, a gene specific for riboflavin biosynthesis was isolated from a eukaryote, Saccharomyces cerevisiae, in order to establish a recombinant method for producing riboflavin in a eukaryotic producing microorganism.
However, when the production of substrates for enzymes specifically involved in riboflavin biosynthesis is insufficient, this type of recombinant production method has no or very limited success for riboflavin production. I only give you.

Hanson (Hanson AM, 1967, in Microbial Technology, Peppler, HJ, pp222-250
(New York) found in 1967 that the addition of the amino acid glycine increased riboflavin formation in Ashbya gossypii. However, this type of method has the disadvantage that glycine is a very expensive raw material. For this reason, it was necessary to optimize riboflavin production by producing mutants.

German patent publication 19545468-A1 describes a method for the microbial production of riboflavin in which the citrate lyase activity or isocitrate lyase-gene expression of the riboflavin-producing microorganism is increased. In addition, DE198
Known from 40709A1 are unicellular or multicellular organisms, in particular microorganisms, for the biotechnological production of riboflavin. This microorganism has the ability to synthesize riboflavin
Wild-type ATC of at least Ashbya gossypii species without external addition of glycine
It is superior in that it shows altered glycine metabolism, similar to the ability of C10892 to synthesize riboflavin.

However, even compared to this method, there is a need for further optimization of riboflavin production.

The object of the present invention is therefore to provide unicellular or multicellular organisms, advantageously microorganisms, for the biotechnological production of riboflavin, which allows further optimization of riboflavin formation. In particular, it should provide organisms that enable production that is economical over the prior art. In particular, this organism should be capable of increased riboflavin formation compared to conventional organisms.

This problem is solved by a unicellular or multicellular organism in which the enzymatic activity for NAD (P) H-formation is higher than the enzymatic activity for NAD (P) H-formation of wild-type ATCC 10895 of Ashbya gossypii species.

The purpose of accelerated intracellular NAD (P) H-supply is either by increasing the activity of the NAD (P) H-forming enzyme or by decreasing the activity of the enzyme consuming NAD (P) H, or This can be achieved by changing the specificity. This can be achieved by known methods of strain improvement of organisms. Thus, in the simplest case, the corresponding strains can be produced by selection by screening routine in microbiology. Similarly, it is possible to use mutations and subsequent selection. On this occasion,
Mutations can be performed by either chemical mutagenesis or physical mutagenesis. A further method is selection and mutation, and subsequent recombination. Finally, the organism according to the invention can be produced by genetic engineering.

According to the present invention, an organism is transformed so that it produces intracellular NAD (P) H in greater amounts than required to maintain its metabolism. This increase in intracellular NAD (P) H-production can be achieved according to the invention by producing an organism in which the enzymatic activity of isocitrate dehydrogenase is increased. This can be achieved, for example, by changing the catalytic center, by increasing the substrate conversion or by the elimination of the action of the enzyme inhibitor. Increased enzymatic activity of isocitrate dehydrogenase can be triggered by enhancing enzymatic synthesis, for example by genetic manipulation or by blocking factors that suppress enzymatic biosynthesis.

Isocitrate dehydrogenase activity can be increased according to the invention, advantageously by mutation of the isocitrate dehydrogenase gene. Mutations of this kind can be performed in a classical manner randomly, for example by UV-irradiation or by mutagenesis chemicals, or specifically by genetic engineering techniques, such as deletions, insertions and / or
Alternatively, it can be induced by nucleotide substitution.

Isocitrate dehydrogenase-gene expression can be achieved by the integration of isocitrate dehydrogenase gene copies and / or by enhancing the regulatory factors that positively influence isocitrate dehydrogenase gene expression. The strengthening of the regulatory elements preferably takes place at the time of transcription, in particular by increasing the transcription signal. In addition, enhanced translation is possible, in which case, for example, the stability of m-RNA is improved.

To increase the gene copy number, for example, the isocitrate dehydrogenase gene is preferably attached to the isocitrate dehydrogenase gene (zugeordnete)
It can be constructed in a gene construct or in a vector containing regulatory gene sequences, in particular regulatory genes that enhance gene expression. Subsequently, the gene construct containing the isocitrate dehydrogenase gene is transformed into a riboflavin-producing microorganism.

According to the present invention, overexpression of isocitrate dehydrogenase can be achieved by replacing the promoter. Alternatively, a high enzymatic activity can be achieved here by incorporating a gene copy or by exchanging the promoter. However, it is also possible in the same way to achieve the desired change in enzyme activity by simultaneously exchanging promoters and incorporating gene copies.

Altering the isocitrate dehydrogenase gene promoted NAD (P
) Leads to H-formation and at the same time an unexpectedly high rise in riboflavin formation, which was heretofore unattainable.

Isocitrate dehydrogenase is preferably isolated from microorganisms, particularly preferably fungi. In that case, fungi of the genus Ashbya are also advantageous. Most advantageous is the Ashbya gossypii species.

However, for the isolation of the gene, all other organisms in which the cell has the sequence for the formation of isocitrate dehydrogenase, plant and animal cells can be mentioned. Isolation of the gene is also carried out by homologous and heterologous complementarity of mutants deleted in the isocitrate dehydrogenase gene or by PCR with heterologous probing or heterologous primers. For subcloning, the insert of the complementary plasmid can be subsequently minimized by suitable steps with restriction enzymes. After sequencing and identification of the putative gene, fully compatible subcloning by PCR is performed. The plasmid with the fragment thus obtained as an insert is integrated into an isocitrate dehydrogenase gene deletion mutant, which is tested for the function of the isocitrate dehydrogenase gene. Finally, the functional construct is used for transformation of riboflavin producers.

After isolation and sequencing, an isocitrate dehydrogenase gene having a nucleotide sequence encoding the described amino acid sequence or allelic variants thereof is obtained. Allelic variants include in particular derivatives obtained by deleting, inserting and substituting nucleotides from the corresponding sequences, the isocitrate dehydrogenase activity being retained. The corresponding sequence is described in Figure 2b, nucleotides 1-1262.

The isocitrate dehydrogenase gene is specifically defined by nucleotide-66 according to FIG.
1 to -1 nucleotide sequence promoter, or DNA- having substantially the same action
It has the sequence upstream. Thus, a promoter that differs from a promoter having said nucleotides by insertion and / or deletion by one or more nucleotide substitutions without affecting the function or effect of the promoter is present, for example upstream of this gene. Good. Furthermore, the promoter can either be enhanced in its effect by a change in its sequence or it can be completely replaced by a valid promoter.

The isocitrate dehydrogenase gene may be provided with other regulatory gene sequences or genes that increase the activity of the isocitrate dehydrogenase gene in particular. Thus, for example, the isocitrate dehydrogenase gene has a so-called "
An "enhancer" may be attached, which enhancer acts on elevated isocitrate dehydrogenase expression via an improved interaction between RNA-polymerase and DNA.

One or more DNA-sequences upstream and / or in the isocitrate dehydrogenase gene with or without a promoter upstream or with or without a regulatory gene It may be located downstream and thus the gene is contained in the genetic construct. Cloning of the isocitrate dehydrogenase gene yields a plasmid or vector containing the isocitrate dehydrogenase gene and suitable for transforming riboflavin producers. The cells obtained by transformation are in a replicable form of the gene, ie on the chromosome with an additional copy (where the gene copy is inserted by homologous recombination at any position in the genome) and / or on a plasmid or vector. Have.

The unicellular or multicellular organisms obtained according to the invention may be cells which can be optionally used in biotechnological methods. This can include, for example, fungi, yeasts, bacteria and plant and animal cells. Preferred according to the invention are transformed cells from fungi, particularly preferably from fungi of the genus Abyssia. On this occasion,
The Ashbya gossypii species are particularly advantageous.

The present application is described in detail below with reference to examples, but the present application is not limited to these examples: The gene for isocitrate dehydrogenase (IDP3) is cloned by PCR and then sequenced (sequence See FIG. 11). Geneticin resistance to geneticin-partial deletion of gene by exchange mutagenesis in gene (Fig. 1)
Was confirmed by Southern blot (Figure 2). This disruption, or the breakdown of a gene in the fungal genome, leads to the fungus being unable to form the isocitrate dehydrogenase encoded by this gene. FIG. 3 shows the reduction of enzyme activity in the disrupted strain AgΔDP3b compared to wild type ATCC10895. In the production of peroxisomes it could be shown that this enzyme is localized in organelles (Fig. 10). The enzyme activity in wild-type-peroxisomes is clearly measurable, whereas no activity is seen in the disrupted strain peroxisomes.

Gene disruption leads to a clear reduction in vitamin formation compared to the parent strain (Fig. 4).
. On the other hand, when the gene is inserted in additional copies and under control of the strong TEF-promoter on the plasmid (Fig. 6) into acibia cells, a clear increase in enzyme activity and riboflavin formation can be measured. Yes (Fig. 5).

FIG. 7 shows that NADPH is required as a reducing agent in two of the three selectable reaction pathways in the metabolism of unsaturated fatty acids. The 2,4-dienoyl-CoA-reductase contained therein is also localized in peroxisomes in Aciba cells (FIG. 8). Disruption of the IDP3-gene reduces cell growth for linoleic acid or linolenic acid. This can also be measured (Fig. 9). It has been shown that the significance of IDP3 for cellular metabolism is in NADPH-formation.

Regarding the sequence: The nucleotide sequence of the AgIDP3-gene encoding the peroxisomal NADP-specific isocitrate-dehydrogenase from Ashbya gossypii and the amino acid sequence derived therefrom.

[Sequence list]

[Brief description of drawings]

FIG. 1 shows the structure of vector pIDPkan for gene exchange of chromosomal AgIDP3-gene for gene copies inactivated by deletion and insertion of G418 ^R -gene.

FIG. 2: Southern blot-analysis of partial deletions and simultaneous insertion of a geneticin resistance-cassette at the AgIDP-locus. Hybridization of genomic SphI-cleaved DNA with a digoxigenin labeled probe.

[Fig. 3] NADP- of wild type Ashibiya, mutant Ag.ΔIDP3b, and AgIDP-overexpressing Ag.pAGIDP3a and AgDPpAGIDP3b.
Comparison of enzyme activity for growth of specific ICDH on complete glucose medium.

FIG. 4 Comparison of growth and riboflavin formation of soybean wild-type and mutant Ag.ΔIDP3b in complete soybean oil medium.

FIG. 5: Ashibiya wild type and AgIDP3− in culture on a soybean oil complete medium.
Comparison of growth, riboflavin formation, and NADP-specific ICDH of overexpressors Ag pAGIDP3a and Ag pAGIDP3b.

FIG. 6: AgIDP3-under the control of TEF-promoter and TEF-terminator.
A plasmid for overexpression of a gene. The introduction of the SphI-cleavage site requires a change in the nucleotide encoding the second amino acid. A conservative substitution of the amino acid glycine for leucine was made.

FIG. 7. Degradation pathway of unsaturated fatty acids with double bonds at even (A) and odd (B, C) C atoms in peroxisomes by Henke et al. (1998).

FIG. 8: Separation of organelles isolated from acibia-wild type in a Percoll-gradient: for the degradation of the marker enzymes catalase (peroxisome) and fumarase (mitochondria), NAD- and NADP-specific ICDH and unsaturated fatty acids. 2,4-dienoyl -CoA- reductase and delta ³ required, delta ² - enoyl -CoA- activity of isomerase [U / ml].

FIG. 9: Ashbya wild type, mutant Ag.ΔIDP3a and Ag.ΔIDP3b and overexpressors Ag.pAGIDP3a and A. on various fatty acids.
Comparison of radial growth of g.pAGIDP3b (A: 18: 1cis9; b: 18:
2cis9,12; C: 18: 3cis9,12,15).

FIG. 10: Distribution of enzymes catalase and ICDH in Percoll-concentration gradient by centrifugation of organelles from mycelium of wild type (A) and mutant Ag.ΔIDP3b (B).

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] September 13, 2001 (2001.9.13)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12R 1: 645 C12P 25/00 C12N 15/00 ZNAA // (C12P 25/00 5/00 A C12R 1: 645) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD) , SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU , ZA, ZW (72) Inventor Henning Alt Hofer Germany Limburger Hof Mainstrasse 12 (72) Inventor Oscar Zelder Federal Republic of Germany Speyer Rothmarkt Strasse 27 (72) Inventor Jose Ereleverta Val Spain Salamanca Grillo 11 4 d (72) Inventor Maria Angeles Santos Garcia Spain Salamancase / Esqueras 1-5 (72) Inventor Hermann Zaam Germany Federal Republic of Eurich Wendelinstrasse 71 (72) Inventor Klaus-Peter Staman Germany Jürich Wilhelmstraße 20 (72) Inventor Enes Mating Germany Jurich Wiesenstraße 3 F Term (reference) 4B024 AA01 AA03 AA05 BA08 BA80 CA02 DA11 FA01 FA02 FA13 GA25 HA01 4B064 CC24 DA05 CA01 DA05 DA24 4B065 AA58X AA58Y AB01 AC14 BA02 BA16 BD50 CA28 CA41 CA44

Claims (21)

[Claims] 1. Bioengineering riboflavin, characterized in that the enzymatic activity for NAD (P) H-formation is higher than the enzymatic activity for NAD (P) H-formation of wild-type ATCC 10895 of Ashbya gossypii species. Unicellular or multicellular organisms for producing, in particular microorganisms. 2. A unicellular or multicellular organism according to claim 1, which has an increased isocitrate dehydrogenase activity. 3. The unicellular or multicellular organism according to claim 1 or 2, which is a fungus. 4. The unicellular or multicellular organism according to any one of claims 1 to 3, which is a fungus of the genus Ashbya. 5. The unicellular or multicellular organism according to any one of claims 1 to 4, which is a fungus of Ashbya gossypii species. 6. An isocitrate dehydrogenase gene having a nucleotide sequence encoding the amino acid sequence shown in FIG. 11 and an allelic variant thereof. 7. The isocitrate dehydrogenase gene according to claim 6, which has the nucleotide sequence of nucleotides 1-1262 according to FIG. 11 or a DNA sequence which acts essentially the same. 8. The isocitrate dehydrogenase gene according to claim 6, which has an upstream promoter of the nucleotide sequence having nucleotides -661 to -1 according to FIG. 11 or a DNA sequence having substantially the same action. 9. The isocitrate dehydrogenase gene according to any one of claims 6 to 8, which has a regulatory gene sequence attached thereto. 10. A gene structure containing the isocitrate dehydrogenase gene according to any one of claims 6 to 9. 11. A vector containing the isocitrate dehydrogenase gene according to any one of claims 6 to 9 or the gene construct according to claim 10. 12. A transformant for producing riboflavin, which contains the isocitrate dehydrogenase gene according to any one of claims 6 to 9 or the gene structure according to claim 10 in a replicable form. 13. The transformed organism according to claim 12, which contains the vector according to claim 11. 14. A method for producing riboflavin, which comprises using the organism according to any one of claims 1 to 5. 15. The enzyme activity for NAD (P) H-formation is changed so as to be higher than the enzyme activity for NAD (P) H-formation of wild-type ATCC 10895 of Ashbya gossypii species. , A process for producing riboflavin-producing single-cell or multicellular organisms. 16. The method according to claim 15, wherein the organism is changed by a genetic engineering method. 17. The method according to claim 15 or 16, wherein the organism is changed by replacing the promoter and / or increasing the gene copy number. 18. The method according to any one of claims 15 to 17, wherein an enzyme having an increased activity due to a change in an endogenous isocitrate dehydrogenase-gene is produced. 19. A method according to claims 1 to 5 and 12 for producing riboflavin.
Use of the organism according to any one of (1) and (13). 20. The isocitrate dehydrogenase gene according to any one of claims 6 to 9 and claim 10 for producing the organism according to any one of claims 1 to 5 and 12 and 13. Use of the gene structure of. 21. Use of the vector according to claim 11 for producing the organism according to any one of claims 1 to 5 and 12 and 13.